论文部分内容阅读
为建立一套适用于刺槐EST-SSR标记的PCR反应体系,采用L16(45)正交试验设计结合单因素试验,对刺槐EST-SSR PCR反应体系中的5个主要因素:引物、DNA模板、dNTPs、Mg2+和Taq DNA聚合酶进行优化试验。结果表明:在20μL反应体系中,各反应因素的最佳水平为0.5μmol/L引物、30 ng模板DNA、0.15 mmol/L dNTP、0.5 mmol/L Mg2+、1.0 UTaq DNA聚合酶。利用随机挑选的3个刺槐优良无性系品种和7对EST-SSR引物对优化体系进行验证,结果均能获得稳定、清晰的条带,证明该体系稳定可靠。此优化体系的建立为刺槐EST-SSR标记的开发以及利用EST-SSR标记深入研究和利用中国刺槐种质资源奠定基础。
In order to establish a set of PCR reaction system suitable for EST-SSR marker of locust, five major factors in EST-SSR PCR reaction system of locust locust were studied by L16 (45) orthogonal design and single-factor experiment: primer, DNA template, dNTPs, Mg2 + and Taq DNA polymerase. The results showed that the optimum reaction conditions were 0.5 μmol / L primer, 30 ng template DNA, 0.15 mmol / L dNTP, 0.5 mmol / L Mg2 + and 1.0 UTaq DNA polymerase in 20 μL reaction system. Three randomly selected Robinia pseudoacacia clones and seven pairs of EST-SSR primers were used to validate the optimized system. Stable and clear bands were obtained and the results showed that the system was stable and reliable. The establishment of this optimized system laid the foundation for the development of EST-SSR markers and the EST-SSR markers for in-depth study and utilization of Chinese locust tree germplasm resources.